Formulation of Sugar Solutions for Continuous Ultracentrifugation for Virus Purification

ABSTRACT

The present invention provides a method for purification of a virus or virus antigen comprising providing a virus preparation and centrifugation of said virus preparation in a gradient of a sugar established by the addition of two or more buffered sugar layers of different concentration. The method leads to higher yields and reduces unwanted aggregation of the virus or virus antigen by increasing the volume of the peak pool.

This application claims the benefit of priority to U.S. ProvisionalApplication No. 60/927,692, filed May 4, 2007, the contents of which areincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of virus purification.

BACKGROUND OF THE INVENTION

Viruses, either those occurring in nature, or recombinant versionsthereof, are used for vaccination and in the field of gene therapy. Itis possible for many viruses or virus-like particles to safely andefficiently propagate in host cells. Several publications describe thepurification of viruses from host cells, mostly concentrating on the useof specific chromatographic matrices for purification of the virus froma host cell lysate (see, e.g. U.S. Pat. No. 6,008,036). Other methods asdescribed, for example, in U.S. Pat. No. 6,048,537 employ continuoussucrose gradient centrifugation, which delivers products with lessantigen purity and requires further purification steps bycentrifugation. Such methods also suffer from antigen aggregation, whichcan lead to a loss of viral antigen, or inhibit viral inactivationsteps.

Viral aggregation can also inhibit the yield of chromatographicprocesses, which are time and cost intensive and difficult to adapt to alarge scale production. It is therefore a goal of the present inventionto provide a method to purify viruses, in particular from host cellsamples, that is simple but still is capable of providing whole virusantigen fractions in high purity with reduced viral antigen aggregation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Microphotograph of PMVHs purified with the conventional (FIG. 1a) and the inventive (FIG. 1 b) ultracentrifugation procedures.

SUMMARY OF THE INVENTION

The present invention provides a method for purification of a virus orvirus antigen comprising providing a virus preparation andcentrifugation of said virus preparation in a gradient of a sugarestablished by a first layer A of the sugar in a concentration of asucrose equivalent of 34% to 50% (w/w %) and a second layer B of thesugar in a concentration of a sucrose equivalent of 50% to 65% (w/w %)sucrose, in an aqueous buffer, with one buffer component being a buffercomponent having a pKa value between 6.0 and 9.4, and collecting saidvirus or virus antigen from the centrifugate, i.e. the product of thecentrifugation process.

One aspect of the invention is to provide a method for purification of avirus or virus antigen. The method involves providing a solutioncontaining a sugar gradient established by centrifuging at least a firstbuffered sugar solution and at least a second buffered sugar solution.The concentration of sugar in the first buffered sugar solution has asucrose equivalent of 35% to 50% (w/w %), or in certain embodiments, 34%to 46% (w/w %). The concentration of sugar second buffered sugarsolution has a sucrose equivalent of 50% to 65% (w/w %), or in certainembodiments, 50% to 60% (w/w %). Once the sugar gradient is established,a virus preparation is added to the sugar gradient. The viruspreparation and sugar gradient are then centrifuged to obtain a peakpool, and the peak pool is extract to obtain the virus or virus antigen

DETAILED DESCRIPTION OF THE INVENTION

During purification of a virus or viral antigen, the virus or the viralantigen is concentrated by centrifugation, allowing a separation fromcell culture media components and/or host cells and host cellcomponents. The inventive method can minimize virus aggregation andvirus loss at the centrifugation step of purification.

In the methods of the invention, a first sugar solution in aphysiological buffer is loaded generally vertically into a continuousultracentrifugation apparatus to form a horizontal sugar solution layerA, and then a second, higher concentration sugar solution in the same ordifferent physiological buffer is loaded into the apparatus to form ahorizontal sugar solution layer B. The apparatus is then activated,creating a sugar gradient that is most concentrated on outer wall of theapparatus, and that becomes less concentrated towards the center of theapparatus. A virus containing harvest solution from a cell culture isthen loaded into the apparatus, and the virus particles migrate to alocation in the sugar gradient where their density is equivalent to thedensity of the gradient. When the sugar is sucrose, a typical densityrange where this equilibration occurs is 36%-48% (w/w %) sucrose. Oncethe apparatus is halted, the gradient shifts to a horizontal position,and the portion of the gradient which contains the virus particles (or“peak pool”) is withdrawn from the apparatus.

One aspect of this invention is the surprising discovery that byutilizing a twolayer solution approach, the volume of the peak pool canbe increased as compared to conventional methods utilizing a singleconcentration of sucrose solution in water to form a sucrose gradient.This surprising effect is obtained even if sucrose is used and themaximum sucrose concentration in the gradient is the same as used in theconventional method. By optimizing the concentrations and amounts of thetwo sucrose solution layers A and B, the peak pool volume can beincreased, for example, by two-fold. This increased peak pool volumereduces the aggregation of whole virus particles, or antigen byreduction of their respective concentrations.

Continuous ultracentrifugation processes are useful for purification ofvirus preparations. Highly concentrated solutions of sugar (e.g.,sucrose or sorbitol) or salt (e.g. CsCl₂ or NaBr) can be used togenerate gradients by ultracentrifugation. However, sugar solutionswithout any additives have a low concentration of electrolytes and nobuffer capacity. Thus, one aspect of the invention is the realizationthat virus preparations in sugar solutions, such as sucrose solutions,have a strong tendency to form aggregates, dependent on theconcentration of the virus particles, which can cause problems in thefurther processing for vaccine production. The inventive methods caneffectively minimize these problems using sugar solutions (e.g., sucrosesolutions at 55% w/w) with the addition of salt at physiologicalconcentrations (e.g., NaCl at about 4-8 g/kg) and with the addition of abuffer (e.g., Tris, approximately 10-20 mmol/kg, followed by pHadjustment) to achieve physiological electrolyte and pH conditions inthe gradient. In certain embodiments, different sugar solutions areapplied to build the gradient in the ultracentrifuge. As an example, 200mL of a 55% w/w solution and 800 mL of a 42% w/w solution are loaded tothe ultracentrifuge. The smaller amount of higher concentration sucrosesolution ensures that at the end of the ultracentrifugation the maximumsucrose concentration remains above 45% and the larger amount of 42%sucrose results in a more gradual gradient in the range of about 40%where the virus peak maximum typically occurs. By these methods of theinvention, the volume of the peak pool can be increased, and thereforethe virus particle suspension is diluted and results in lessaggregation. Also, the diffusion of molecules or particles between thesupernatant which is loaded to the ultracentrifuge and the gradient ismodified by using such sucrose formulations.

By way of example, a density gradient formed using approximately 42% and55% (w/w %) sucrose solution in 20 mmol/kg Tris-buffer was found to beparticularly useful for the influenza virus but can also be easilyadapted for other viruses. Tris (trishydroxymethylaminomethane) is auseful buffer for pH ranges between 6.5 and 9.7. Although sucrose isused in this particular example of an embodiment of the invention, othersugars suitable for gradient centrifugation may be used. In addition,other buffer compounds, in particular organic buffer compounds, such asamines, can be used. Preferably the buffer has a pKa value above 6, 6.2,6.4, 6.6, 6.8, 7.2, 7.4, 7.6, 7.8 or 8.0 and below 9.4, 9.2, 9.0, 8.8,8.6, 8.4 or 8.2. Suitable buffers comprise for example HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), ACES(N-(2-Acetamido)-2-aminoethanesulfonic acid), bis-tris-methane or-propane, CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) or CAPSO,PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)), phosphate buffer, orany other buffer used in the field of biochemistry. The buffer ispreferably chemically inert to biomolecules.

The centrifugation process can generate a peak pool fraction in whichthe virus particle is dispersed under physiological conditions withregard to electrolyte and pH conditions. The concentration of wholevirus antigen in the peak pool fraction can be modified by addingdifferent amounts of high and low concentration sugar solutions todefine the gradient and therefore the volume of the peak fraction.Additionally, the volume of the peak fraction is also increased by theaddition of salt and a suitable buffer (e.g. Tris-buffered saline (TBS))alone, leading to a significantly higher yield of antigen and protein.This effect might be due to an unexpected improved mobility of proteinsand virus antigen into a sugar gradient containing physiologicalconcentrations of salt and a physiological pH. Different ultracentrifugeconditions (e.g., with and without preclarifier and differentgravitational forces) clearly showed that a significant yield increaseand a reduced aggregation of virus particles can be achieved by thesemodifications of the sucrose gradient.

Preferably the addition of electrolytes is greater than 50 mOsm/kg, 100mOsm/kg, 150 mOsm/kg, 200 mOsm/kg or 250 mOsm/kg and less than 500mOsm/kg, 450 mOsm/kg, 400 mOsm/kg or 350 mOsm/kg, preferably about 300mOsm/kg. It should be noted that the amount of electrolyte is calculatedas equivalent to the osmolality of aqueous solutions without the van'tHoffs factor correction. Moreover, the contribution of thegradient-forming substance (e.g., sucrose) is not used for thecalculation. For example, the osmolality from electrolytes is calculatedas follows: 8 g NaCl/kg/58.44 g/mol=136.9 mmol/kg×2 (ions/mol)=273.8mOsm/kg+20 mM/kg TRIS×2 ions/mol=40 mOsm/kg; Total (NaCl+TRIS) 313.8mOsm/kg). The pH value can be in physiological range, preferably between5.5 and 9.5, more preferably between 5.5 and 8.5, especially preferredabove 5.5, 6, 6.5 or 7 and below 9.5, 9.0, 8.5, 8 or 7.5, in particularbetween 7.0 and 7.5 or between 7.0 and 7.4.

In a particularly preferred embodiment, the sugar used to create adensity gradient is sucrose. However, this invention also contemplatesthe use of other sugars, including alcohol sugars, a non-limitingexample of which is sorbitol. Furthermore, the invention contemplatesthe use of hydrogenated sugars, linear sugars, modified sugars or anyother sugar, provided that the sugar has a solubility in watersufficient to produce the solutions with the densities specified herein.Combinations of sugars (i.e., two or more sugars), either in the same ordifferent layers, can also be used to generate the gradients describedherein, provided that the corresponding sugar solutions that constitutethose layers have the densities specified herein.

In preferred embodiments, the volume of the gradient forming preparationis between 5% to 100%, preferably above 5%, 10% 15%, 20%, 25%, 30%, 32%,35%, 40%, 45% or 50% or less than 100%, 90%, 80%; 75%, 70%, 65%, 60%,55%, 50%, 48%, 45%, 40%, 35%, 30% or 25%, of the effective volume of theultracentrifugation rotor or device applying centrifugal forces to thesample. In other embodiments, the volume of gradient forming preparationis between 1% to 75% of the effective volume of the ultracentrifugationrotor or device applying centrifugal forces to the sample.

In preferred embodiments, the loading of the rotor containing thegradient forming material is carried out in a continuously ordiscontinuously repeated mode (i.e., either continuousultracentrifugation or batch ultracentrifugation), where the loadedvolume is preferably higher than the void volume of the rotor (totalvolume minus gradient volume), preferably more than 1×, 2×, 3×, 5×, 10×,20×, 30×, 40×, 50×, 60×, 80× or less than 300×, 250×, 200×, 150×, 120×,100×, 80×. In certain particularly preferred embodiments, continuousultracentrifugation is used.

In a particularly preferred embodiment, the loaded volume is between 20L to 50 L per 600 mL to 1000 mL void volume (1600 mL rotor volume minus600 mL to 1000 mL void volume), which is about a 20× to 80× ratio. Inanother embodiment, the loaded volume is between 40 L to 100 L per 1200mL to 2000 mL void volume (3200 mL rotor volume minus 1200 mL to 2000 mLvoid volume, which is about a 20× to 80× ratio).

During centrifugation a continuous gradient of the sugar is established.In a further embodiment a fraction of the centrifugate is collectedabove a sucrose concentration of 24% (equivalent to about 1.10 kg/l),26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44% or 46% (w/w %)(equivalent to about 1.22 kg/l) and less than 66% (equivalent to about1.32 kg/l), 63%, 60%, 58%, 56%, 54%, 52%, 50%, 48%, 46% 44% or 42% (w/w%) (equivalent to about 1.19 kg/l). Normally, the viral productaccumulates in this range. Preferably, a fraction of the centrifugate iscollected between a sucrose concentration of about 30% and 54% (w/w %),more pre-ferred between 36% and 48% (w/w %) sucrose. Fractions collectedfrom gradients formed from other sugars might have differentconcentrations (w/w %), but equivalent densities. The densities of thecollected fraction may be above 1.10 kg/l, 1.12 kg/l, 1.14 kg/l, 1.16kg/l or 1.18 kg/l, 1.20 kg/l, 1.22 kg/l and below 1.32 kg/l, 1.30 kg/l,1.28 kg/l, 1.26 kg/l, 1.24 kg/l, 1.22 kg/l or 1.20 kg/l. In particularlypreferred embodiments, the densities may be between 1.13 and 1.25 kg/l,more preferred between 1.16 and 1.22 kg/l.

In certain embodiments, the concentration of buffer or buffer component(e.g., TRIS buffered saline) is in between 2 mmol/kg to 50 mmol/kg,preferably 5 mmol/kg to 40 mmol/kg, 10 mmol/kg to 40 mmol/kg, morepreferably between 10 mmol/kg to 30 mmol/kg, more preferably between 18mmol/kg to 25 mmol/kg, and is most preferred to be 20 mmol/kg. Thebuffer is preferably aqueous with less than 5% water-miscible organicsolvents, more preferably less than 2% organic solvents, and is mostpreferably free of organic solvents. The buffer has a concentration ofmore than 2 mmol/kg, 5 mmol/kg, 10 mmol/kg, 12 mmol/kg, 14 mmol/kg, 15mmol/kg, 17 mmol/kg, 18 mmol/kg, 19 mmol/kg or 20 mmol/kg or less than50 mmol/kg, 40 mmol/kg, 35 mmol/kg, 30 mmol/kg, 25 mmol/kg, 23 mmol/kgor 21 mmol/kg.

The centrifugation can be performed preferably with a centrifugal forceof at least 20,000 g, more preferably 30,000 g, more preferably 50,000g, more preferably 70,000 g, most preferably 90,000 g, but in otherembodiments, the centrifugal force is less than 200,000 g, less than150,000 g, less than 120,000 g, less than 100,000 g, or less than 90,000g.

In particular preferred embodiments, the volume ratio of the initiallayer A to the initial layer B (i.e. before application of the viruspreparation) is between 20:1 to 1:1, preferably between 10:1 to 1.5:1,more preferably between 8:1 to 2:1 even more preferably between 6:1 to3:1, and most preferably 4:1. The ratio can be less than 20:1, 15:1,10:1, 8:1, 5:1, 4:1, 3:1 or greater than 1:2, 1:1, 1.5:1, 2:1, 3:1 or4:1.

In a particularly preferred embodiment, the sugar-containingcentrifugation fluid is two-layered (i.e., comprising the sugar layers Aand B of different concentrations prior to addition of the viruspreparation, below the filler layer of liquid in the ultracentrifuge).Of course during centrifugation new concentration gradients or densitygradients may form. However, this invention also contemplatescentrifugation fluids with more than two layers. For example, in certainnon-limiting embodiments, the centrifugation fluid is comprised of 3, 4,5, 6, 7 or even 8 different layers. Each layer may have the same bufferas the other layers, or the buffers of each layer may be independentlychosen.

In particularly preferred embodiments, the method of preparation doesnot comprise a preclarification step. However, if desired,preclarification may be carried out in a pre-clarification chamber ofthe ultracentrifugation apparatus.

In further embodiments layer A comprises between 40% to 44% (w/w %)sucrose, preferably from 41% to 43% (w/w %) sucrose. The sucroseconcentration may be above 35% (equivalent to about 1.15 kg/l), 36%,37%, 38%, 39%, 40%, 41% or 42% (w/w %) (equivalent to about 1.19 kg/l)or below 50% (equivalent to about 1.23 kg/l), 49%, 48%, 47%, 46%, 45%,44%, 43% or 42% (w/w %) (equivalent to about 1.19 kg/l). Layer A formedfrom another sugar might have a different concentration on aweight/weight basis, but will have a density that falls within thedensity ranges specified herein.

In certain embodiments, layer B comprises a sucrose solution having aconcentration between 50% to 65% (w/w %), preferably between 52% to 58%(w/w %), and more preferably between 54% to 56% (w/w %). The sucroseconcentration (w/w %) may be above 50% (equivalent to about 1.23 kg/l),51%, 52%, 53%, 54% or 55% (equivalent to about 1.26 kg/l) or below 66%(equivalent to about 1.32 kg/l), 64%, 62%, 61%, 60%, 59%, 58%, 57%, 56%or 55% (equivalent to about 1.26 kg/l). Layer B formed from other sugarsmight have a different concentration on a weight/weight basis, but willhave a density that falls within the density ranges specified herein.

In a preferred embodiment of the present invention, the virus is anorthomyxovirus, in particular an influenza virus, preferably selectedfrom influenza A and B. Non-limiting examples of other virusescontemplated by the invention include viruses selected from the group ofRNA virus families such as Reoviridae, Picornaviridae, Caliciviridae,Togaviridae, Arenaviridae, Retroviridae, Flaviviridae, Orthomyxoviridae,Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae,Coronaviridae, Astroviridae, Bornaviridae, and DNA virus families suchas Adenoviridae, Papovaviridae, Parvoviridae, Herpesviridae, Poxviridae,Hepadnaviridae. In certain preferred embodiments, the virus is selectedfrom the group consisting of Influenza A/Panama/2007/99, A/NewCaledonia/20/99, and B/Shangdong/7/97.

In some embodiments of the invention, the virus harvest is prepared fromcells inoculated with the virus. The virus can be produced in any cellssuitable for the production of viruses. Preferably, the cells are of ananimal cell culture or cell line. Such cells may be from a specifictissue or embryonic cells such as embryonic eggs. The animal ispreferably a mammal or a bird. In various embodiments of the invention,the cells are avian, canine, rodent, or primate cells. In specificembodiments, the cells are epithelial cells, in particular preferredkidney epithelial cells, such as Vero cells of an African green monkey.

The virus harvest applied to the ultracentrifugation apparatus may bethe whole cell culture, or cell culture supernatant obtained afterseparating cells and/or portions thereof from the cell culture. In someembodiments of the invention, cells in the cell culture are allowed tosettle in the culture vessel prior to drawing off a cell culturesupernatant. In other embodiments of the invention, the cells may belysed by chemical or mechanical methods, non-limiting examples of whichinclude hypo or hypertonic lysis, detergent treatment, sonication, andmechanical disruption.

Preferably, the virus is inactivated or fragmented (e.g., as describedin WO 05/11800) prior to or after the centrifugation. Additionally, avaccine of the virus may be pre-pared by methods known in the art. Avaccine is an immunogenic composition of an antigenic substance, i.e.the (non-infections) virus, its hull, particles or its antigens, whichan be used to immunize an animal, e.g. a mammal such as a human, or abird.

The present invention is further illustrated by the following exampleswithout being limited thereto.

EXAMPLES

During purification of influenza virus antigen, the monovalent harvest(MVH) is concentrated by ultracentrifugation. A continuous flowcentrifugation procedure can be applied for the manufacture of the Verocell culture grown viral vaccine based on a sucrose gradient formedusing a 50% (w/w %) sucrose solution in water. The centrifuge rotormodel used was equipped with a preclarifier. A number of fermentationoptimization approaches like change of the Vero cell culture medium bysupplementing soy hydrolysate or elevated temperature conditions in theearly phase of Influenza virus replication resulted in a more robustprocess as well as increased antigen yields. Therefore it turned outthat the 50% (w/w %) sucrose/water gradient did not allow to achieve thedesired recovery due to limitations in the antigen binding capacity. Inaddition, the PMVHs (purified MVH) of virus strains New Caledonia,Panama and Shangdong showed unexpected antigen aggregation at thisparticular production step. Several attempts were made to minimize virusaggregation and virus loss at the centrifugation step. TRIS bufferedsaline instead of water was used to dissolve the sucrose for thegradient material. Furthermore the modification of using two gradientforming solutions with different densities was introduced to increasethe peak pool volume. In addition, ultracentrifugation withoutpreclarifier but with increased g-forces turned out to be a valuabletool for yield improvement. To prove this concept, results from previouspurifications with standard (50% sucrose in water, with preclarifier at20,000 rpm=90,000 g) and new conditions (42% and 55% sucrose (w/w %), 20mmol/kg TRIS, 8 g/kg NaCl, without preclarifier at 35,000 rpm=90,000 g,with a harvest of PMVH from 48%-36% sucrose fraction) were evaluated.

Example 1 Materials and Methods

A continuous flow ultracentrifuge CC40S with a C40CTS rotor (rotorvolume 1,600 mL) or an Alfa Wassermann ultracentrifuge RK6 with anequivalent rotor was used. Sucrose gradient solutions were loaded to therotor and then accelerated to the rotational speed of 20,000 to 35,000rpm. After continuous loading with the inactivated harvest, the rotorwas flushed with buffer to remove residual protein, which had notentered the gradient. After flushing, the rotor was decelerated and theultracentrifugation was stopped, allowing the gradient to shift from theradial to axial direction of the rotor. Elution and fractionation wascarried according to sucrose concentrations. A Coriolis type densitydetection unit and a UV 254 nm detection unit were used to monitor thesucrose and protein concentration.

Samples of the purified inactivated harvests were measured for totalprotein concentration, HA-SRD and Vero Antigen by ELISA according tostandardized procedures to quantify yield and purity from theultracentrifugation experiments.

Example 2 Initial Experiments with a TBS-Sucrose Gradient

A number of small scale purification runs with A/Panama/2007/99monovalent virus harvests indicated that the use of a sucrose gradientproduced from a mixture of 50% w/w sucrose with 50% w/w Tris bufferedsaline (20 mmol/kg TRIS, 8 g/kg NaCl) (final concentration 10 mmol/kgTRIS, 4 g/kg NaCl) had several advantages over the standardsucrose/water system. A laboratory ultracentrifuge model RK-6 was usedto purify 25 liter and 50 liter MVH aliquots under different conditions.An overview of the parameter setup and the results for purification runswith the sucrose/water and sucrose/TBS system is given in Table 1.

TABLE 1 Comparison of Influenza A/Panama/2007/99 antigen yield and PMVHappearance. Sucrose gradient purified virus from ultracentrifugationexperiments with preclarifier at 30,000 g. HA- SRD/ Peak MVH proteinPool Antigen yield Purifica- Conditions/ Load ratio Volume (mg antigen/PMVH tion Run Setup (liter) (mg/mg) (mL) liter harvest) (appearance) 1Sucrose/water 25 0.51 333 1.2 aggregation 800 mL 2 Sucrose/TBS 25 0.34440 2.0 reduced 800 mL aggregation 3 Sucrose/TBS 50 0.26 492 1.4 reduced800 mL aggregation

From Table 1 it can be concluded that sucrose gradients in TBS haveseveral advantages over the standard gradients in the sucrose/watersystem. For Purification Run 1 a yield of 1.2 mg antigen/L harvest couldbe achieved, the antigen fraction (PMVH) showed unexpected virusaggregation. The purification experiments (run 2) with sucrose in TBSgave increased antigen yields compared to reference run 1. For the TBSpreparations a significant reduction in the aggregate fraction could beobserved. Although the TBS sucrose gradient has several advantages overthe standard system (sucrose/water), a loss in antigen yield wasobserved at higher loading (50 liters compared to 25 liters) of thegradient (run 3).

In both cases of using TBS instead of water, the peak pool volumeincreased, which is indicative for more diffusion between the loadedharvest and the gradient containing a buffer substance and otherelectrolytes.

Example 3 Development of a Two-Step Sucrose/TBS Gradient

This example shows an exemplary embodiment in which a sucrose gradientwas modified to increase the volume of the peak pool fraction withoutchanging the limits for fractionation. A sucrose gradient with a reducedconcentration of 42% (w/w %) was used, which resulted in a less steepsucrose gradient eluted from the ultracentrifuge. In order to ensure asufficiently high sucrose concentration in the gradient, a moreconcentrated sucrose/TBS solution of 50% (w/w %) was loaded subsequentto the 42% (w/w %) solution, which, as a higher concentration solution,formed a “cushion” to prevent from sedimentation of antigen on the wallof the rotor. Table 2 shows purification runs using such a two-stepgradient with 900 mL of a less concentrated sucrose/TBS solution (42%w/w sucrose, 11.7 mmol/kg TRIS, 4.7 g/kg NaCl) and 100 mL of a moreconcentrated sucrose/TBS solution (50% w/w, 10 mmol/kg TRIS, 4 g/kgNaCl), compared to a purification run using a gradient formed only witha 50% sucrose/TBS solution, applied in different volumes (viz., 600,800, and 1000 mL). The tests were performed with pre-clarifier at 20,000rpm (RCF appr. 30,000 g).

By applying the different amounts and concentration of the sucrose/TBSsolutions the yield and purity of the material were not influencedsignificantly. However, the use of the two-step gradient built up by themixture of 42%/50% (w/w %) sucrose/TBS resulted in an significantincrease of the peak pool volume, which leads to better purificationconditions by reduction of the antigen concentration to avoidoverloading of the peak pool fraction.

TABLE 2 Comparison of Product yield and Antigen purity of inactivatedharvests of A/Panama/2007/99 purified by ultracentrifugation withdifferent amounts of Sucrose/TBS 50% (w/w %) vs. a two Step Sucrose/TBSgradient (50%/42% (w/w %)). HA- Antigen yield Peak Pool Purifica-SRD/protein (mg antigen/liter Volume tion Run Conditions/Setup ratio(mg/mg) harvest) (mL) 1 Sucrose/TBS 50% 600 mL 0.58 1.3 346 2Sucrose/TBS 50% 800 mL 0.58 1.3 325 3 Sucrose/TBS 50% 1000 mL 0.45 1.1338 4 Sucrose/TBS 42% 900 mL + 0.53 1.5 713 Sucrose/TBS 50% 100 mL

Example 4 Implementation of Higher Relative Centrifugal Forces withoutPreclarifier

Higher relative centrifugal forces for the ultracentrifugation wereinvestigated to increase the antigen yield, however loss of material inthe preclarifier had to be expected under such conditions. Theultracentrifugation rotors can be run with or without preclarifier,therefore a comparison of 20,000 rpm (appr. 30,000 g) with preclarifierand 35,000 rpm (appr. 90,000 g) in a two step sucrose/TBS gradient (100mL of 50% w/w sucrose, final concentration 10 mmol/kg TRIS, 4 g/kg NaCland 900 mL of 39% w/w sucrose, final concentration 12.2 mmol/kg TRIS,4.9 g/kg NaCl) was carried out. In order to assess the potential loss ofantigen, the preclarifier and the rotor wall were swabbed with bufferand analyzed for antigen losses. The results of this experiment areshown in Table 3. The antigen yield could be increased by over 60% from1.7 mg HA-SRD/L harvest to 2.8 mg HA-SRD/L harvest. The main part of themissing antigen could be swabbed from the preclarifier, whereas onlyabout 5% of the antigen was lost on the rotor wall of theultracentrifuge. The SRD/Protein ratio is only slightly reduced byomitting the preclarification prior to loading the harvest onto thesucrose/TBS gradient.

TABLE 3 Comparison of product yield and losses and antigen purity byultracentrifugation of inactivated harvest of A/Panama/2007/99 withdifferent relative centrifugal forces with and without preclarifier.Loss Rotor Loss in Pre- HA- Antigen yield wall clarifier Purifica-Conditions/ SRD/protein (mg antigen/ [mg SRD/L [mg SRD/L tion Run Setupratio (mg/mg) liter harvest) Harvest] Harvest] 1 20,000 rpm 0.63 1.7 0.11.4 w/Preclarifier 2 35,000 rpm 0.52 2.8 0.1 N/A w/o Preclarifier

Example 5 Modification of the TRIS and NaCl Concentration in the SucroseGradients

Initial formulations were carried out by preparing a 50% w/w sucrose/TBSgradient by mixing of 50% (weight) sucrose to 50% (weight) TBS buffer(20 mmol/kg TRIS, 8 g/kg NaCl) resulting in final concentrations of 10mmol/kg TRIS and 4 g/kg NaCl. Subsequently, less concentratedformulations 42%-39% sucrose/TBS solutions were prepared with higheramounts of TBS, resulting in lower concentrations of sucrose butcorrespondingly higher concentrations of TRIS and NaCl (see previousexamples). More concentrated solutions like, for example, 55% (w/w/)sucrose in TBS had therefore lower concentrations of TRIS and NaCl.

In order to standardize the concentration of TRIS and NaCl in suchpreparations a new formulation was designed where 42%-55% weight sucrosewas added to the mixing container, 20 mmol/kg (2.42 g/kg) TRIS and 8g/kg NaCl were added and filled with water to 100% weight. Suchpreparations then had significantly (about 2×) higher concentrations ofTRIS and NaCl compared to the formulations used in previous examples.Also refractometric measurements of such formulations resulted in about2° (1°-3°) Brix higher refractometric results, due to the lesser amountof water, which was replaced by the addition of TRIS and NaCl beforefilling to the final weight of 100%.

In an experiment, two different versions of two-step sucrose/TBSgradients were compared. Gradients were built by adding 200 mL of thehigher concentrated sucrose/TBS solution to create a more robust, highdensity sucrose cushion in the gradient, after loading with 800 mL of42% sucrose/TBS.

The results of the different sucrose/TBS preparations in anultracentrifuge experiment are shown in Table 4. No significantdifferences could be observed with regard to yield or purity whendifferent TRIS and NaCl concentrations were used.

TABLE 4 Comparison of Product yield and Antigen purity byUltracentrifugation of inactivated harvest of A/Panama/2007/99 withdifferent Sucrose/TBS preparations with different TRIS and NaClconcentrations. HA- Antigen yield Purifica- SRD/protein (mgantigen/liter tion Run Conditions/Setup ratio (mg/mg) harvest) 1 42%sucrose in TBS 0.42 5.0 (12.2 mmol/kg TRIS/ 4.9 g/kg NaCl) 55% sucrosein TBS (8.9 mmol/kg TRIS/ 3.6 g/kg NaCl) 2 42% sucrose in TBS 0.39 4.8(20 mmol/kg TRIS/ 8 g/kg NaCl) 55% sucrose in TBS (20 mmol/kg TRIS/ 8g/kg NaCl)

Example 6 Loading Capacity of the Rotor and Investigation of the LoadingFlow Rate

Different harvest volumes of three different strains were investigatedto assess the capacity of the optimized ultracentrifugation procedureand to investigate potential scale-up issues in manufacturing, whenhigher harvest volumes have to be applied to the ultracentrifugationprocedure. Inactivated harvests were loaded with 15-17 L and 45-51 L onthe 1600 mL rotor.

In this experiment, the same modified version of the two-stepsucrose/TBS gradient was applied as in the previous example 5. First,the concentration of the higher density solution was increased to 55%w/w and a higher volume of 200 mL was used to create a more robust highdensity sucrose cushion in the gradient, after loading with 800 mL of42% sucrose/TBS. Final TRIS concentration in the gradient was increasedto 20 mmol/kg and the final NaCl concentration was increased to 8 g/kgin both sucrose/TBS solutions. Ultracentrifugation conditions were35,000 rpm (90,000 g) without preclarifier, and fractionation wascarried out between 48% to 36% sucrose.

The results of the loading capacity experiment are shown in Table 5.

With the inventive gradient and the higher centrifugation force withoutpreclarifier only minor differences in yield of about 3% to 6% atdifferent harvest loading volumes could be observed. This is in contrastto the limited loading capacity demonstrated in Example 2/Table 1, wherefor strain A/Panama/2007/99 an increase of the harvest volume from 25liter to 50 liter resulted in a decrease in yield of about 30%. Thisimproved yield can be explained by the combination of increased relativecentrifugal forces and the improvement of the gradient profile and peakpool volume due to the two-step sucrose/TBS gradient.

TABLE 5 Comparison of Product yield and Antigen purity byUltracentrifugation with different Loading Volumes. HA- Antigen yieldPurifica- Strain/ SRD/protein (mg antigen/liter tion Run Harvest Volumeloaded ratio (mg/mg) harvest) 1 A/New Caledonia/20/99 0.37 1.6 45 liter2 A/New Caledonia/20/99 0.45 1.7 15 liter 3 A/Panama/2007/99 0.36 1.9 51liter 4 A/Panama/2007/99 0.44 2.0 17 liter 5 B/Shangdong/7/97 0.44 6.045 liter 6 B/Shangdong/7/97 0.51 6.2 15 liter

Example 7 Comparison of the Inventive and ConventionalUltracentrifugation Procedures

The combined use of:

-   -   a) 90,000 g (35,000 rpm) without preclarifier (instead of 30,000        g (20,000 rpm) with preclarifier)    -   b) a sucrose gradient in TBS (instead of sucrose in water) with        a final concentration of 20 mmol/kg TRIS and 8 g/kg NaCl    -   c) forming such a gradient with two different sucrose        concentrations (200 mL 55%+800 mL 42%) to increase the peak pool        fraction volume (instead of 800 mL of 50% sucrose)        was compared with a previous, conventional ultracentrifugation        procedure (Table 6).

TABLE 6 Conditions and setup of the manufacturing and small scale (1:2scale down version) Purification unit operation at standard (50% sucrosein water) and new conditions. Virus Load Fraction- Method GradientSucrose (l/hr) RPM ation standard Sucrose/ 800 mL 50% 12.5 20.000 acc.to water UV signal (max. 50%-34%) new Sucrose/ 800 mL 42% 12.5 35.000*48%-36% TBS 20 mM 200 mL 55% *without preclarifier

Three comparative experiments were carried out with the 2002/2003strains (viz., A/New Caledonia/20/99, A/Panama/2007/99 andB/Shangdong/7/97). The peak pool fractionation limits for these runswere defined with 48% to 36% (w/w %) sucrose. The results are shown inExample 8 to Example 10.

The results from purification runs with Influenza New Caledonia, Panamaand Shangdong under standard (50% sucrose/water) and modified conditions(sucrose/TBS gradient) were compared on the basis of yield and productquality parameters.

Example 8 Purification of Influenza Strain A/New Caledonia/20/99

Purification experiments with conditions according to the standardprocedure and the new sucrose/TBS gradient parameters according to Table6 were carried out with New Caledonia MVH. In Table 7 a comparison ofthe results from purification runs is given.

TABLE 7 Comparison of A/New Caledonia/20/99 antigen yield, HA/proteinratio and Vero-protein impurity. Sucrose gradient purified virus withthe conventional 2001/2002 method vs modified TBS gradient. Antigenyield (mg antigen/liter HA/total Vero-protein/ Purification Run harvest)protein ratio HA ratio Standard 2.7 0.41 0.06 New (TBS 3.4 0.60 0.07gradient)From the data in Table 7, it can be concluded that a significant processimprovement was achieved with the new sucrose/TBS gradient purificationconditions for Influenza strain A/New Caledonia/20/99. A significantincrease in virus yield from 2.7 to 3.4 mg antigen per liter MVH wasdemonstrated for this particular strain. The virus antigen qualitymeasured as HA/total protein ratio and Vero-protein/HA ratio shows thatthis increase in doses per liter did not compromise antigen quality. Aslight increase in the HA/total protein from 0.41 to 0.60 was achievedwith the new sucrose gradient conditions. For the host cell protein/HAratio no significant difference could be detected. Under the sucrose/TBSgradient conditions for PMVH production, aggregation of strain A/NewCaledonia/20/99 was slightly reduced. This effect could be confirmed bymicroscopic observation (see, e.g., FIG. 1A and FIG. 1B). At 400×magnification the stronger, formation of aggregates is clearly visiblewhen applying the previous sucrose/water gradient version (FIG. 1A),whereas despite increased yields, the peak pool fraction derived fromthe optimized ultracentrifugation procedure appears to be significantlymore homogenous (FIG. 1B).

Example 9 Purification of Influenza Strain A/Panama/2007/99

Purification experiments with conditions according to the standardprocedure and the new sucrose/TBS gradient parameters according to Table6 were carried out with Panama MVH. In Table 8 a comparison of theresults from the purification runs is given.

TABLE 8 Comparison of A/Panama/2007/99 antigen yield, HA/protein ratioand Vero-protein impurity. Sucrose gradient purified virus with thestandard method vs the inventive TBS gradient method. Antigen yield (mgantigen/liter HA/total Vero-protein/ Purification Run harvest) proteinratio HA ratio standard 2.0 1.47 0.06 new (TBS 5.5 0.77 0.07 gradient)

From the data in Table 8 it can be concluded that a significant processimprovement was achieved with the new sucrose/TBS gradient purificationconditions for Influenza strain Panama. A significant increase in virusyield from 2.0 to 5.5 mg antigen per liter MVH was demonstrated for thisparticular strain by applying the new sucrose/TBS gradient conditions. AHA/total protein ratio of 0.77 was achieved, whereas for the PMVHproduced under standard conditions the calculated ratio was 1.47. Due tothe fact that no significant difference can be seen for theVero-protein/HA ratio of PMVHs produced with both procedures, it isassumed that aggregation of the Panama antigen (as seen in amicrophotograph comparison of Panama PMVHs (not shown)) may be the causefor the unusual HA/total protein ration of 1.47. For both purificationprocedures, the virus antigen quality measured as HA/total protein ratioand Vero-protein/HA ratio is acceptable. But, under the TBS sucrosegradient conditions for PMVH production, aggregation of strain Panamacould be significantly reduced using the sucrose/TBS gradient.

Example 10 Purification of Strain B/Shangdong/7/97

Purification experiments with conditions according to the standardprocedure and the new sucrose/TBS gradient parameters according to Table6 were carried out with Shangdong MVH. In Table 9 a comparison of theresults from purification runs is given.

TABLE 9 Comparison of B/Shangdong/7/97antigen yield, HA/protein ratioand Vero-protein impurity. Sucrose gradient purified virus with thestandard method vs new TBS gradient method. Antigen yield (mgantigen/liter HA/total Vero-protein/ Purification Run harvest) proteinratio HA ratio standard 2.2 0.55 0.10 new (TBS 5.1 0.34 0.20 gradient)

From the data in Table 9, it can be concluded that a significant processimprovement was achieved with the inventive sucrose/TBS gradientpurification conditions for Influenza strain B/Shangdong/7/97. Asignificant increase in virus yield from 2.2 to 5.1 mg antigen per literMVH was demonstrated for this particular strain. The reduced HA/totalprotein ratio of 0.34 for the sucrose/TBS purified MVH lies within thespecification range of the Influenza PMVHs. Further improvements of theB/Shangdong/7/97 virus replication phase allowed to establish a moreconsistent production process by changing the trypsin dosage profile.Under the TBS sucrose gradient conditions for PMVH production,aggregation of the B/Shangdong/7/97strain was significantly reduced asconfirmed by a microphotograph (not shown) of Shangdong PMVHs purifiedwith the standard versus TBS-conditions.

In the exemplary large and small scale experiments provided herein, itwas demonstrated that the inventive sucrose gradient allows efficientloading of the monovalent harvests. There are many advantages of usingthe inventive methods described herein, including 1) increased virusyield of at least 25%, 2) a strain dependant HA/total protein ratio of0.34 to 0.77, 3) a strain dependant host cell protein content of 2% to7% and 4) minimized virus aggregation in the sucrose peak pool (PMVH).

1. A method for purification of a virus or virus antigen comprisingproviding a gradient-forming solution containing a sugar gradient bycentrifuging at least a first buffered sugar solution and at least asecond buffered sugar solution, wherein the concentration of sugar inthe first buffered sugar solution has a sucrose equivalent between 35%to 50% (w/w %) and the concentration of sugar in the second bufferedsugar solution has a sucrose equivalent between 50% to 65% (w/w %), andwherein the second buffered sugar solution has a higher density than thefirst buffered sugar solution; adding a virus preparation to the sugargradient, centrifuging the virus preparation and sugar gradient toobtain a peak pool, and extracting the peak pool to obtain the virus orvirus antigen.
 2. The method of claim 1, wherein the volume of thegradient-forming solution is between 5% to 100% of the volume of theultracentrifugation rotor.
 3. The method of claim 1, wherein is the peakpool has a density between 1.13 kg/l to 1.25 kg/l.
 4. The method ofclaim 1, wherein the peak pool is collected with a sucrose equivalentbetween 30% and 54% (w/w %) sucrose.
 5. The method of claim 1, whereinat least one of said buffers has a concentration of between 5 mM to 50mM.
 6. The method of claim 5, wherein said at least one buffer has aconcentration of between 15 mM to 30 mM.
 7. The method of claim 6,wherein said at least one buffer has a concentration of between 18 mM to25 mM.
 8. The method of claim 1, wherein the step of centrifuging thevirus preparation is performed with a relative centrifugation of atleast 20,000 g.
 9. The method of claim 8, wherein said relativecentrifugation force is at least 30,000 g.
 10. The method of claim 9,wherein said relative centrifugation force is at least 90,000 g.
 11. Themethod of claim 1, wherein said volume ratio of said first bufferedsugar solution to said at least a second buffered sugar solution isbetween 20:1 to 1:1.
 12. The method of claim 11, wherein said volumeratio is between 10:1 to 1.5:1.
 13. The method of claim 12, wherein saidvolume ratio is between 8:1 to 2:1.
 14. The method of claim 13, whereinsaid volume ratio is between 6:1 to 3:1.
 15. The method of claim 1,wherein said solution containing a sugar gradient comprises two layers.16. The method of claim 1, wherein said preparation does not comprise apreclarifier.
 17. The method of claim 1, wherein said at least a firstbuffered sugar solution comprises a sugar in a concentration range from40% to 44% (w/w %) sucrose equivalent.
 18. The method of claim 17,wherein said at least a first buffered sugar solution comprises a sugarin a concentration range of 41% to 43% (w/w %) sucrose equivalent. 19.The method of claim 1, wherein said at least a second buffered sugarsolution comprises a sugar in a concentration range from 52% to 58% (w/w%) sucrose equivalent.
 20. The method of claim 19, wherein said at leasta second buffered sugar solution comprises a sugar from 54% to 56%sucrose equivalent.
 21. The method of claim 1, wherein said virus is anorthomyxovirus.
 22. The method of claim 21, wherein said virus is aninfluenza virus.
 23. The method of claim 1, wherein said viruspreparation comprises cells inoculated with the virus.
 24. The method ofclaim 23, wherein said cells are of an animal cell culture or cell line.25. The method of claim 23, wherein said cells are epithelial cells. 26.The method of claim 25, wherein said cells are kidney epithelial cells.27. The method of claim 26, wherein said cells are Vero cells.
 28. Themethod of claim 1, wherein said virus is inactivated.
 29. The method ofclaim 1, wherein said virus is fragmented.
 30. The method of claim 1,wherein the buffer component is an amine.
 31. The method of claim 31,wherein the buffer component is TRIS.
 32. The method of claim 1, whereinthe buffer is TRIS-buffered saline.
 33. A method of preparing a vaccineagainst a virus or viral antigen, wherein said virus or viral antigen isobtained using the method according to claim
 1. 34. A method forpurification of a virus or virus antigen comprising providing agradient-forming solution containing a sugar gradient by centrifuging atleast a first buffered sugar solution and at least a second bufferedsugar solution, wherein the density of the first buffered solution is1.15 kg/l to 1.23 kg/l and the density of the second buffered sugarsolution is 1.23 kg/l to 1.32 kg/l, and wherein the second bufferedsugar solution has a higher density than the first buffered sugarsolution; adding a virus preparation to the sugar gradient, centrifugingthe virus preparation and sugar gradient to obtain a peak pool, andextracting the peak pool to obtain the virus or virus antigen.